Bottom Line:
Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible.Fluorescent readouts revealed moderate transfection rates (30-50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell.This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection.

fig03: Analyzing the EGFP-LcA transcellular protein crossover by monitoring enzymatic activity. A: Neuro2A cells were transfected with EGFP-LcA; 6 h after transfection the medium was discarded, cells were washed with PBS, and new complete DMEM was added. B: Western immunoblotting of SNAP25 showing the total enzymatic efficacy of EGFP-LcA at different time points (h). C: The new medium was sampled at intervals. This sampled medium, absent of exogenous DNA, was applied to fresh Neuro2A cells treated with LTX then incubated for 42 h. This medium sampled from previously transfected cells was then centrifuged to separate soluble (supernatant, SN) and suspended (pellet, P) fractions. These fractions were added onto new Neuro2A cells treated with or without Lipofectamine LTX. D: Western immunoblotting of SNAP25 shows enzymatic efficacy within the untransfected Neuro2A cells where the sampled medium was applied and treated with Lipofectamine LTX. The previously sampled medium thus contains the EGFP-LcA protein as detected by enzymatic activity. E: Western immunoblotting of SNAP25 shows that soluble (SN) and suspended membrane bound (P) EGFP-LcA are found freely floating in the medium and can, mediated by Lipofectamine LTX, enter new Neuro2A cells to cleave intracellular SNAP25.

Mentions:
To ascertain whether a much higher percentage of transfection actually occurs but lies below the threshold of detection or whether the EGFP-LcA can escape the confines of these transfected cells to cleave SNAP25 in non-transfected cells we sampled the culture medium at different time points. Figure 3 shows a schematic of this transfection protocol. Figures 3A and B shows the total enzymatic readout within undisturbed EGFP-LcA transfected Neuro2A cells; after 24 h a substantial amount of SNAP25 cleavage can already be observed. This culture medium, which was replaced 6 h after transfection, was then sampled at different time points and administered to new Neuro2A cells that were treated with Lipofectamine LTX only (Fig. 3C). Since the cells were washed 6 h after transfection, we removed the remaining free-floating DNA-lipoplexes that could have been carried over to the second generation of cells. Figure 3D shows that the protease-contaminated culture medium drawn from the first generation of transfected cells as early as 10 h post-wash (16 h post-transfection) can cleave SNAP25 in a second generation of cells. To avoid seeding the wells with pre-cleaved SNAP25 we performed western immunoblotting on the supernatant alone which showed no immunoreactive SNAP25 nor SNAP25Δ9 while resuspended EGFP-LcA transfected cells clearly did (Supplementary Fig. S1). Thus we were not inadvertently transferring EGFP-LcA transfected cells into new wells. Also, as the 0 h post-wash seeding does not show any cleaved-SNAP25 in the second generation of Neuro2A, meaning that the primary cells require synthesis time, this excludes the possibility of inadvertent DNA-Lipoplex carry over. To test this further, we centrifuged the sampled medium (Fig. 3C). As can be seen in Figure 3E, both the soluble (supernatant, SN) and the suspended (pellet, P) fractions contained the protease. And this also shows that the penetration of the proteases is dependent on the presence of the transfection reagent, in this case Lipofectamine LTX. Thus, once the soluble or suspended proteases are released into the culture medium, they can, mediated by the transfection reagent, enter new untransfected cells where they continue their innate functions.

fig03: Analyzing the EGFP-LcA transcellular protein crossover by monitoring enzymatic activity. A: Neuro2A cells were transfected with EGFP-LcA; 6 h after transfection the medium was discarded, cells were washed with PBS, and new complete DMEM was added. B: Western immunoblotting of SNAP25 showing the total enzymatic efficacy of EGFP-LcA at different time points (h). C: The new medium was sampled at intervals. This sampled medium, absent of exogenous DNA, was applied to fresh Neuro2A cells treated with LTX then incubated for 42 h. This medium sampled from previously transfected cells was then centrifuged to separate soluble (supernatant, SN) and suspended (pellet, P) fractions. These fractions were added onto new Neuro2A cells treated with or without Lipofectamine LTX. D: Western immunoblotting of SNAP25 shows enzymatic efficacy within the untransfected Neuro2A cells where the sampled medium was applied and treated with Lipofectamine LTX. The previously sampled medium thus contains the EGFP-LcA protein as detected by enzymatic activity. E: Western immunoblotting of SNAP25 shows that soluble (SN) and suspended membrane bound (P) EGFP-LcA are found freely floating in the medium and can, mediated by Lipofectamine LTX, enter new Neuro2A cells to cleave intracellular SNAP25.

Mentions:
To ascertain whether a much higher percentage of transfection actually occurs but lies below the threshold of detection or whether the EGFP-LcA can escape the confines of these transfected cells to cleave SNAP25 in non-transfected cells we sampled the culture medium at different time points. Figure 3 shows a schematic of this transfection protocol. Figures 3A and B shows the total enzymatic readout within undisturbed EGFP-LcA transfected Neuro2A cells; after 24 h a substantial amount of SNAP25 cleavage can already be observed. This culture medium, which was replaced 6 h after transfection, was then sampled at different time points and administered to new Neuro2A cells that were treated with Lipofectamine LTX only (Fig. 3C). Since the cells were washed 6 h after transfection, we removed the remaining free-floating DNA-lipoplexes that could have been carried over to the second generation of cells. Figure 3D shows that the protease-contaminated culture medium drawn from the first generation of transfected cells as early as 10 h post-wash (16 h post-transfection) can cleave SNAP25 in a second generation of cells. To avoid seeding the wells with pre-cleaved SNAP25 we performed western immunoblotting on the supernatant alone which showed no immunoreactive SNAP25 nor SNAP25Δ9 while resuspended EGFP-LcA transfected cells clearly did (Supplementary Fig. S1). Thus we were not inadvertently transferring EGFP-LcA transfected cells into new wells. Also, as the 0 h post-wash seeding does not show any cleaved-SNAP25 in the second generation of Neuro2A, meaning that the primary cells require synthesis time, this excludes the possibility of inadvertent DNA-Lipoplex carry over. To test this further, we centrifuged the sampled medium (Fig. 3C). As can be seen in Figure 3E, both the soluble (supernatant, SN) and the suspended (pellet, P) fractions contained the protease. And this also shows that the penetration of the proteases is dependent on the presence of the transfection reagent, in this case Lipofectamine LTX. Thus, once the soluble or suspended proteases are released into the culture medium, they can, mediated by the transfection reagent, enter new untransfected cells where they continue their innate functions.

Bottom Line:
Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible.Fluorescent readouts revealed moderate transfection rates (30-50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell.This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection.